In vitro preservation of living animal cells and compounds suitable for use in the preservation of living animal cells

ABSTRACT

The invention is directed to compounds of Formula (I) 
                         
or salts thereof. The invention is also directed to use of the compounds of formula (I) in the in vitro preservation of living animal cells. The living cells can be isolated cells, such as stem cells, or groups of cells such as tissue or an organ.

RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §120 and is aContinuation-in-Part of the International PCT Application Serial No.PCT/GB2008/050931, filed Oct. 10, 2008, which claims the benefit andpriority of Great Britain Patent Application Serial No. 0719751.0, filedOct. 10, 2007. The contents of both applications are incorporated hereinby reference in their entirety.

FIELD OF THE INVENTION

This invention is concerned with in vitro preservation of living animalcells and compounds suitable for use in the preservation of livinganimal cells. In particular, though not exclusively, the presentinvention concerns in vitro preservation of stem cells and compoundssuitable for use in preserving stem cells.

BACKGROUND

Once harvested, in order to remain viable and retain theirundifferentiated state, stem cells must be preserved prior to use formedical or research purposes.

It is well known that stem cells can be stored indefinitely if stored atthe temperature of liquid nitrogen (−196° C.). However, it is welldocumented that the process of freezing may cause irreparable damage tothe cells and as such, various cryopreservative agents can be added tothe cell suspension prior to the freezing process. US2005/0106554discloses methods and compositions for the cryopreservation ofpluripotent cells, in particular human embryonic stem cells. The methodsdisclosed in US2005/0106554 are shown to exhibit an increase in cellviability and a decrease in cell differentiation when compared withconventional methods. The cryopreservation method disclosed inUS2005/0106554 comprises encapsulation of the cells between two layersof a solid support matrix, adding a cryopreservative to thematrix-cell-matrix composition, and cooling said composition to atemperature sufficient to cryopreserve the cells. US2005/0106554discloses a carbohydrate-based medium, preferably trehalose, as asuitable cryopreservative. WO2005/118785 discloses methods for thecryopreservation of stem cells, wherein said method includes performingice nucleation on a cell suspension prior to the reduction oftemperature sufficient to allow long term storage of the stem cells.WO2005/118785 further discloses that the cell suspension may or may notcontain any exogenous biological cryoprotectant, such as serum.

A number of non-cryogenic methods of stem cell preservation are alsoknown. In such methods, stem cells are generally stored as a celldispersion in an aqueous solution containing tissue cell culture growthmedia. Often a preservative compound is included in such aqueoussolutions so as to reduce the rate at which cell viability decreases. Ina typical example, U.S. Pat. No. 5,912,174 discloses a method of storinga population of mammalian cells capable of duplication anddifferentiation by suspending said population of cells in an aqueousmixture containing gelatin. Preferred mixtures contain standard tissuecell culture growth media such as RMPI or Eagle's media. Optionally,cell-specific growth factor may also be added to preserve cell viabilityand, for optimum storage life, the storage temperature should bemaintained between 0 and 4° C. Further, EP-A-1057405 discloses the useof an aqueous storage liquid comprising polyphenol and a storage liquidselected from Euro-Collin's solution, UW solution, serum and antibioticsolution for the in vitro freeze-free preservation of, inter alia, astem cell, tissue or organ for transplantation. The polyphenolsdisclosed in EP-A-1057405 include catechins such as epigallocatechin,tannic acid, proanto-dianisidine, resorcinol, hydroquinone, pyrogallol,phloroglucinol, eugenol and quercetin. There is no suggestion inEP-A-1057405 to the effect that any of the disclosed compounds maypreserve the viability of undifferentiated cells. EP1627565 discloses amedium for storing biological samples in a refrigerated, frozen orvitrified state, comprising a balanced salt solution and4-thioderivative of flavon-3-ol. WO 2006/019366 discloses a culturesystem developed for the culturing of human embryonic stem cells. Theculture condition includes culture of the cells in an atmosphere havingminimal oxygen, and may include the use of an antioxidant.

The structures of various natural and synthetic flavonoid compounds areknown. For example, Heterocycles, Vol. 34, no. 6, pages 1213-1225(Fukai, et al.) discloses the use of ¹H-NMR in order to identifyisoprenoid substituted flavanols. The structures of various flavonoidsare also proposed.

Tetrahedron, Vol. 58, no. 18, pages 2619-2622 (Sutthivaiyakit, et al.)discloses the purification of various flavonoids from the chloroformextract of dried leaves of M. denticulata.

The potential therapeutic use of certain flavonoid compounds asantioxidants for the treatment of patients having a disease or disorderinvolving oxidative damage, such as cancer, heart disease, neurologicaldisorders, auto-immune disorders, ischemia-reperfusion injury, diabeticcomplications, septic shock, hepatitis, atherosclerosis andcomplications arising from HIV or Hepatitis B is known from. WO2004/007475. WO 2004/007475 also discloses the potential application ofthe flavonoid compounds in sunscreen compositions and skincarecompositions. In addition, WO 2004/007475 discloses the potential use ofthe flavonoid compounds as foodstuff stabilizers, where the ability ofthe compounds to combat free radicals is considered to be of utility inpreventing or delaying the deterioration in food quality during storage.However, whilst WO 2004/007475 discloses the use of the flavonoidcompounds for the in vivo therapeutic treatment of living matter and forthe in vitro stabilizing treatment of dead matter, there is nodisclosure or suggestion that the flavonoid compounds would be usefulfor in vitro preservation of living matter.

J. Agric. Food Chem., Vol. 48, pages 3876-3884 (Miranda et al.),disclosed test of certain flavanoid compounds to inhibit in vitrooxidation of human low density lipoprotein. British Journal of Cancer,2003, Vol. 89(2), pages 357-362, and Vol. 89(11), pages 2140-2146,disclose the use of monoHER as providing protection againstdoxorubicin-induced inflammatory effects in vitro and againstdoxorubicin-induced cardiotoxicity in vitro, based on protection ofhuman umbilical cord vascular endothelial cells and neonatal rat cardiacmycocytes, respectively. Both cell types are fully differentiated. FreeRadical Biology & Medicine, 2002, Vol. 32(7), pages 596-604, disclosethe testing of certain flavonoids for protecting primary cultures of ratcortical cells against oxidative stress. The cell types are fullydifferentiated. Bio. & Pharm. Bull., 2001, Vol. 24(12) pages 1373-1379,disclose in vitro protective effect of mixtures of extracts from certainplants, including some flavonoids, against oxidative stress on humanskin cells. The cell types are fully differentiated. Bioorganic &Medicinal Chemistry, Vol. 12, no. 9, pages 2079-2098 (Bennett, et al.)discloses potentially therapeutic antioxidants wherein C₆-C₁₂ alkylchains are attached to the A-ring of either a3,3′,4′,5′-tetrahydroxyflavone or a 3,2′,4′,5′-tetrahydroxyflavone headgroup. J. Agric. Food Chem., Vol. 52, no. 6, pages 1514-1519 (Kajiya, etal.) disclosed tests of the antibacterial activity of certain flavonoidcompounds. JP 2002 255810 A discloses catechin derivatives which possessimproved antimicrobial properties in comparison to naturally occurringcatechins. Journal of Pharmacy and Pharmacology, Vol. 55, no. 1, pages131-142 (Kessler, et al.) and J. Chem. Soc. Vol. 6, pages 1215-1222(Dangles, et al.) both disclose the biochemical evaluation of the effectof quercertin and quercertin derivatives such as its 3-O-glycoside rutinon lipid peroxidation. Both Kessler, et al. and Dangles, et al. suggestthat flavonoids can act both as antioxidants and pro-oxidants undercertain conditions.

WO 2008/062184, published after the priority date of the presentapplication, discloses various flavonoid-type compounds which functionas fluorescent probes and antioxidants and may be useful indiscriminating healthy and stressed cells.

The object of the present invention is to provide an improved method forthe in vitro preservation of living animal cells. In particular, it isan object of the present invention to provide an improved method for thein vitro preservation of a mammalian cell, tissue or organ for researchor medical purposes e.g. transplantation. More preferred, it is anobject of the present invention to provide an improved method for the invitro preservation of stem cells.

THE INVENTION

The invention in its various aspects is as set out in the accompanyingclaims.

In a first aspect, the present invention provides a method for in vitropreservation of living animal cells, said method comprising contactingsaid living animal cells with a compound of Formula I or a salt thereof:

wherein:

-   A) R₁₂ and R₂₆ each independently represent —OH or a glycosidic    functional group; R₁₀, R₁₁, R₁₃, and R₁₄ each independently    represent H, —OH, nitro, halogen, amino, amido, cyano, carboxyl,    sulphonyl, a glycosidic functional group, C₁₋₆ alkoxy-,    hydroxy-C₁₋₆alkyl-, C₁₋₆ alkoxy-C₁₋₆ alkyl-, or a saturated or    unsaturated C₁₋₆ hydrocarbon chain which may be substituted with one    or more of nitro, halogen, amino, amido, cyano, carboxyl, sulphonyl,    hydroxyl, ketone or aldehyde groups; and wherein ring B comprises no    more than one glycosidic functional group;-   B) either a):    -   R₂₀ represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon        chain;    -   R₂₁:    -   i) represents H;    -   ii) together with R₂₂ provides a second bond between C¹ and C².    -   iii) when X is —NR₁— and R₁ is not H or C₁₋₆alkyl, together with        R₁ provides a second bond between C¹ and N;    -   R₂₂:    -   i) represents H;    -   ii) together with R₂₃ forms ═O; or    -   iii) together with R₂, provides a second bond between C¹ and C²;    -   R₂₃:    -   i) represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon        chain; or    -   ii) together with R₂₂ forms ═O;    -   wherein at least one of R₂₀ and R₂₃ is a C₂₋₃₀ saturated or        unsaturated hydrocarbon chain;-    or b):    -   R₂₀, R₂₁, R₂₂, and R₂₃ form part of a 5, 6 or 7 membered        unsaturated-ring including C¹ and C², which ring is substituted        with a group which is a C₂₋₃₀ saturated or unsaturated        hydrocarbon chain, which ring is optionally and independently        further substituted with one or more groups selected from nitro,        halogen, amino, amido, cyano, carboxyl, sulphonyl, hydroxyl,        ketone, aldehyde and saturated or unsaturated C₂₋₁₅ hydrocarbon        chain, which C₂₋₁₅ hydrocarbon chain may be substituted with one        or more of nitro, halogen, amino, amido, cyano, carboxyl,        sulphonyl, hydroxyl, ketone, aldehyde or nitrone groups;-   C) n is 0 or 1, wherein when n is 0, either i) R₂₇ and R₂₈ represent    H or ii) R₂₇ together with R₂₈ provide a second bond between C⁴ and    C⁵; or when n is 1, either i) R₂₄ and R₂₅ together form ═O and R₂₇    and R₂₈ represent H or R₂₇ together with R₂₈ provide a second bond    between C⁴ and C⁵, or ii) R₂₄ and R₂₅ represent H and R₂₇ and R₂₈    represent H or R₂₇ together with R₂₈ provide a second bond between    C⁴ and C⁵ or iii) R₂₄ represents H, R₂₅ together with R₂₇ provide a    second bond between C³ and C⁴, R₂₈ represents —OH and X is —O—;-   D) X is —O—, —S— or —NR₁—, wherein R₁ represents i) H or C₁₋₆alkyl,    or ii) together with R₂, provides a second bond between C¹ and N;    wherein said C₂₋₃₀ saturated or unsaturated hydrocarbon chain of    R₂₀, R₂₃ and the 5, 6 or 7 membered unsaturated ring is optionally    and independently substituted with one or more groups selected from    C₁₋₆ alkyl, C₁₋₆alkoxy, hydroxy-C₁₋₆ alkyl, Cl, F, Br, I, —CN,    —CO₂H, —CO₂C₁₋₆alkyl, —S(O)₂C₁₋₆alkyl, —S(O)₂phenyl, —SC₁₋₆alkyl,    —NO₂, —OH, —CF₃, —N(R₂)(R₃), —NHC(O)NHC₁₋₆alkyl, —C(O)N(R₂)(R₃),    imine and substituted or unsubstituted triphenylphosphonium; and    wherein one or more available —CH₂— groups present in the C₂₋₃₀    hydrocarbon chain of R₂₀, R₂₃ or the 5, 6 or 7 membered unsaturated    ring is optionally and independently substituted by —O—, —C(O)—,    —S(O)_(p)—, or —N(R₂)— provided always that no two such    substitutions in the resulting chain are consecutive; wherein R₂ and    R₃ each independently represent H or C₁₋₆alkyl, and wherein p is 0    to 2; and    wherein the total number of ═O on ring C is no greater than 1.

In one embodiment, the present invention provides a method of in vitropreservation of living animal cells in a viable non-terminallydifferentiated state, said method comprising contacting livingnon-terminally differentiated animal cells with a compound of Formula Ior a salt thereof:

wherein:

-   A) R₁₂ and R₂₆ each independently represent —OH or a glycosidic    functional group; R₁₀, R₁₁, R₁₃, and R₁₄ each independently    represent H, —OH, nitro, halogen, amino, amido, cyano, carboxyl,    sulphonyl, a glycosidic functional group, C₁₋₆ alkoxy-, hydroxy-C₁₋₆    alkyl-, C₁₋₆ alkoxy-C₁₋₆ alkyl-, or a saturated or unsaturated C₁₋₆    hydrocarbon chain which may be substituted with one or more of    nitro, halogen, amino, amido, cyano, carboxyl, sulphonyl, hydroxyl,    ketone or aldehyde groups; and wherein ring B comprises no more than    one glycosidic functional group;-   B) either a):    -   R₂₀ represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon        chain;    -   R₂₁:    -   i) represents H;    -   ii) together with R₂₂ provides a second bond between C¹ and C²;        or    -   iii) when X is —NR₁— and R₁ is not H or C₁₋₆alkyl, together with        R₁ provides a second bond between C¹ and N;    -   R₂₂:    -   i) represents H;    -   ii) together with R₂₃ forms ═O; or    -   iii) together with R₂, provides a second bond between C¹ and C²;    -   R₂₃:    -   i) represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon        chain;    -   ii) together with R₂₂ forms ═O;    -   wherein at least one of R₂₀ and R₂₃ is a C₂₋₃₀ saturated or        unsaturated hydrocarbon chain;-    or b):    -   R₂₀, R₂₁, R₂₂, and R₂₃ form part of a 5, 6 or 7 membered        unsaturated-ring including C¹ and C², which ring is substituted        with a group which is a C₂₋₃₀ saturated or unsaturated        hydrocarbon chain, which ring is optionally and independently        further substituted with one or more groups selected from nitro,        halogen, amino, amido, cyano, carboxyl, sulphonyl, hydroxyl,        ketone, aldehyde and saturated or unsaturated C₂₋₁₅ hydrocarbon        chain, which C₂₋₁₅ hydrocarbon chain may be substituted with one        or more of nitro, halogen, amino, amido, cyano, carboxyl,        sulphonyl, hydroxyl, ketone, aldehyde or nitrone groups;-   C) n is 0 or 1, wherein when n is 0, either i) R₂₇ and R₂₈ represent    H or ii) R₂₇ together with R₂₈ provide a second bond between C⁴ and    C⁵; or when n is 1, either i) R₂₄ and R₂₅ together form ═O and R₂₇    and R₂₈ represent H or R₂₇ together with R₂₈ provide a second bond    between C⁴ and C⁵, or ii) R₂₄ and R₂₅ represent H and R₂₇ and R₂₈    represent H or R₂₇ together with R₂₈ provide a second bond between    C⁴ and C⁵ or iii) R₂₄ represents H, R₂₅ together with R₂₇ provide a    second bond between C³ and C⁴, R₂₈ represents —OH and X is —O—;-   D) X is —O—, —S— or —NR₁—, wherein R₁ i) represents H or C₁₋₆alkyl,    or ii) together with R₂₁ provides a second bond between C¹ and N;    wherein said C₂₋₃₀ saturated or unsaturated hydrocarbon chain of    R₂₀, R₂₃ and the 5, 6 or 7 membered unsaturated ring is optionally    and independently substituted with one or more groups selected from    C₁₋₆ alkyl, C₁₋₆alkoxy, hydroxy-C₁₋₆ alkyl, Cl, F, Br, I, —CN,    —CO₂H, —CO₂C₁₋₆alkyl, —S(O)₂C₁₋₆alkyl, —S(O)₂phenyl, —SC₁₋₆alkyl,    —NO₂, —OH, —CF₃, —N(R₂)(R₃), —NHC(O)NHC₁₋₆alkyl, —C(O)N(R₂)(R₃),    imine and substituted or unsubstituted triphenylphosphonium; and    wherein one or more available —CH₂— groups present in the C₂₋₃₀    hydrocarbon chain of R₂₀, R₂₃ and the 5, 6 or 7 membered unsaturated    ring is optionally and independently replaced by —O—, —C(O)—,    —S(O)_(p)—, or —N(R₂)— provided always that no two such replacements    in the resulting chain are consecutive; wherein R₂ and R₃ each    independently represent H or C₁₋₆alkyl, and wherein p is 0 to 2; and    wherein the total number of ═O on ring C is no greater than 1.

In another embodiment, the present invention provides a method for invitro preservation of living animal cells wherein said living animalcells are in the form of a tissue or an organ, said method comprisingcontacting said living animal cells with a compound of Formula I or asalt thereof:

wherein:

-   A) R₁₂ and R₂₆ each independently represent —OH or a glycosidic    functional group; R₁₀, R₁₁, R₁₃, and R₁₄ each independently    represent H, —OH, nitro, halogen, amino, amido, cyano, carboxyl,    sulphonyl, a glycosidic functional group, C₁₋₆ alkoxy-, hydroxy-C₁₋₆    alkyl-, C₁₋₆ alkoxy-C₁₋₆ alkyl-, or a saturated or unsaturated C₁₋₆    hydrocarbon chain which may be substituted with one or more of    nitro, halogen, amino, amido, cyano, carboxyl, sulphonyl, hydroxyl,    ketone or aldehyde groups; and wherein ring B comprises no more than    one glycosidic functional group;-   B) either a):    -   R₂₀ represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon        chain;    -   R₂₁:    -   i) represents H;    -   ii) together with R₂₂ provides a second bond between C¹ and C²;        or    -   iii) when X is —NR₁— and R₁ is not H or C₁₋₆alkyl, together with        R₁ provides a second bond between C¹ and N;    -   R₂₂:    -   i) represents H;    -   ii) together with R₂₃ forms ═O; or    -   iii) together with R₂, provides a second bond between C¹ and C²;    -   R₂₃:    -   i) represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon        chain; or    -   ii) together with R₂₂ forms ═O;    -   wherein at least one of R₂₀ and R₂₃ is a C₂₋₃₀ saturated or        unsaturated hydrocarbon chain;-    or b):    -   R₂₀, R₂₁, R₂₂, and R₂₃ form part of a 5, 6 or 7 membered        unsaturated-ring including C¹ and C², which ring is substituted        with a group which is a C₂₋₃₀ saturated or unsaturated        hydrocarbon chain, which ring is optionally and independently        further substituted with one or more groups selected from nitro,        halogen, amino, amido, cyano, carboxyl, sulphonyl, hydroxyl,        ketone, aldehyde and saturated or unsaturated C₂₋₁₅ hydrocarbon        chain, which C₂₋₁₅ hydrocarbon chain may be substituted with one        or more of nitro, halogen, amino, amido, cyano, carboxyl,        sulphonyl, hydroxyl, ketone, aldehyde or nitrone groups;-   C) n is 0 or 1, wherein when n is 0, either i) R₂₇ and R₂₈ represent    H or ii) R₂₇ together with R₂₈ provide a second bond between C⁴ and    C⁵; or when n is 1, either i) R₂₄ and R₂₅ together form ═O and R₂₇    and R₂₈ represent H or R₂₇ together with R₂₈ provide a second bond    between C⁴ and C⁵, or ii) R₂₄ and R₂₅ represent H and R₂₇ and R₂₈    represent H or R₂₇ together with R₂₈ provide a second bond between    C⁴ and C⁵ or iii) R₂₄ represents H, R₂₅ together with R₂₇ provide a    second bond between C³ and C⁴, R₂₈ represents —OH and X is —O—;-   D) X is —O—, —S— or wherein R₁ represents i) H or C₁₋₆alkyl, or ii)    together with R₂₁ provides a second bond between C¹ and N;    wherein said C₂₋₃₀ saturated or unsaturated hydrocarbon chain of    R₂₀, R₂₃ and the 5, 6 or 7 membered unsaturated ring is optionally    and independently substituted with one or more groups selected from    C₁₋₆ alkyl, C₁₋₆alkoxy, hydroxy-C₁₋₆ alkyl, Cl, F, Br, I, —CN,    —CO₂H, —CO₂C₁₋₆alkyl, —S(O)₂C₁₋₆alkyl, —S(O)₂phenyl, —SC₁₋₆alkyl,    —NO₂, —OH, —CF₃, —N(R₂)(R₃), —NHC(O)NHC₁₋₆alkyl, —C(O)N(R₂)(R₃),    imine and substituted or unsubstituted triphenylphosphonium; and    wherein one or more available —CH₂— groups present in the C₂₋₃₀    hydrocarbon chain of R₂₀, R₂₃ or? the 5, 6 or 7 membered unsaturated    ring is optionally and independently substituted by —O—, —C(O)—,    —S(O)_(p)—, or —N(R₂)— provided always that no two such    substitutions in the resulting chain are consecutive; wherein R₂ and    R₃ each independently represent H or C₁₋₆alkyl, and wherein p is 0    to 2; and    wherein the total number of ═O on ring C is no greater than 1.

As well as concerning a method for the in vitro preservation of livinganimal cells, the present invention is also concerned with novelcompounds. Surprisingly, these novel compounds have been found to besuitable for use as preservatives for living animal cells.

Accordingly, in another aspect of the present invention, there isprovided a compound of Formula Ia or a salt thereof:

wherein:

-   A) R₁₂ and R₂₆ each independently represent —OH or a glycosidic    functional group; R₁₀, R₁₁, R₁₃, and R₁₄ each independently    represent H, —OH, nitro, halogen, amino, amido, cyano, carboxyl,    sulphonyl, a glycosidic functional group, C₁₋₆ alkoxy-, hydroxy-C₁₋₆    alkyl-, C₁₋₆ alkoxy-C₁₋₆ alkyl-, or a saturated or unsaturated C₁₋₆    hydrocarbon chain which may be substituted with one or more of    nitro, halogen, amino, amido, cyano, carboxyl, sulphonyl, hydroxyl,    ketone or aldehyde groups; and wherein ring B comprises no more than    one glycosidic functional group;-   B) either a):    -   R₂₀ represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon        chain;    -   R₂₁:    -   i) represents H;    -   ii) together with R₂₂ provides a second bond between C¹ and C²,        or    -   iii) when X is —NR₁— and R₁ is not H or C₁₋₆alkyl, together with        R₁ provides a second bond between C¹ and N;    -   R₂₂:    -   i) represents H;    -   ii) together with R₂₃ forms ═O; or    -   iii) together with R₂₁ provides a second bond between C¹ and C²;    -   R₂₃:    -   i) represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon        chain; or    -   ii) together with R₂₂ forms ═O;    -   wherein at least one of R₂₀ and R₂₃ is a C₂₋₃₀ saturated or        unsaturated hydrocarbon chain;-    or b):    -   R₂₀, R₂₁, R₂₂, and R₂₃ form part of a 5, 6 or 7 membered        unsaturated-ring including C¹ and C², which ring is substituted        with a group which is a C₂₋₃₀ saturated or unsaturated        hydrocarbon chain, which ring is optionally and independently        further substituted with one or more groups selected from nitro,        halogen, amino, amido, cyano, carboxyl, sulphonyl, hydroxyl,        ketone, aldehyde and saturated or unsaturated C₂₋₁₅ hydrocarbon        chain, which C₂₋₃₀ cchydrocarbon chain may be substituted with        one or more of nitro, halogen, amino, amido, cyano, carboxyl,        sulphonyl, ketone, aldehyde or nitrone groups;-   C) n is 0 or 1, wherein when n is 0, either i) R₂₇ and R₂₈ represent    H or ii) R₂₇ together with R₂₈ provide a second bond between C⁴ and    C⁵; or when n is 1, either i) R₂₄ and R₂₅ together form ═O and R₂₇    and R₂₈ represent H or R₂₇ together with R₂₈ provide a second bond    between C⁴ and C⁵, or ii) R₂₄ and R₂₅ represent H and R₂₇ and R₂₈    represent H or R₂₇ together with R₂₈ provide a second bond between    C⁴ and C⁵ or iii) R₂₄ represents H, R₂₅ together with R₂₇ provide a    second bond between C³ and C⁴, R₂₈ represents —OH and X is —O—;-   D) X is —O—, —S— or —NR₁—, wherein R₁ represents i) H or C₁₋₆alkyl,    or ii) together with R₂, provides a second bond between C¹ and N;    wherein said C₂₋₃₀ saturated or unsaturated hydrocarbon chain of    R₂₀, R₂₃ and the 5, 6 or 7 membered unsaturated ring is optionally    and independently substituted with one or more groups selected from    C₁₋₆ alkyl, C₁₋₆alkoxy, hydroxy-C₁₋₆ alkyl, Cl, F, Br, I, —CN,    —CO₂H, —CO₂C₁₋₆alkyl, —S(O)₂C₁₋₆alkyl, —S(O)₂phenyl, —SC₁₋₆alkyl,    —NO₂, —OH, —CF₃, —N(R₂)(R₃), —NHC(O)NHC₁₋₆alkyl, —C(O)N(R₂)(R₃),    imine and substituted or unsubstituted triphenylphosphonium; and    wherein one or more available —CH₂— groups present in the C₂₋₃₀    hydrocarbon chain of R₂₀, R₂₃ or the 5, 6 or 7 membered unsaturated    ring is optionally and independently replaced by —O—, —C(O)—,    —S(O)_(p)—, or —N(R₂)— provided always that the resulting chain    includes a —CH₂— group connecting to C¹, C² or the 5, 6 or 7    membered ring and no two such replacements are consecutive; wherein    R₂ and R₃ each independently represent H or C₁₋₆alkyl, and wherein p    is 0 to 2; and    wherein the total number of ═O on ring C is no greater than 1;    provided that when i) n=1, ii) X represents —O—, iii) R₁₂ represents    —OH, iv) R₂₄ together with R₂₅ represent ═O, v) R₂₀, R₂₁, R₂₂ and    R₂₃ form a benzene ring including C¹ and C², and vi) said benzene    ring is substituted with at least one group which is a C₂₋₃₀    saturated or unsaturated hydrocarbon chain, then:    -   said C₂₋₃₀ saturated or unsaturated hydrocarbon chain is        substituted with one or more groups selected from C₁₋₆alkoxy,        hydroxy-C₁₋₆ alkyl, Cl, F, Br, I, —CN, —CO₂H, sulphonyl,        —CO₂C₁₋₆alkyl, —S(O)₂C₁₋₆alkyl, —S(O)₂phenyl, —SC₁₋₆alkyl, —NO₂,        —OH, —CF₃, —N(R₂)(R₃), —NHC(O)NHC₁₋₆alkyl, —C(O)N(R₂)(R₃), imine        and substituted or unsubstituted triphenylphosphonium; and/or        wherein one or more available —CH₂-groups present in said C₂₋₃₀        hydrocarbon chain is replaced by —O—, —C(O)—, —S(O)_(p)—, or        —N(R₂)—; wherein R₂ and R₃ each independently represent H or        C₁₋₆alkyl, and wherein p is 0 to 2; and/or    -   said benzene ring is substituted with one or more groups        selected from nitro, halogen, amino, amido, cyano, carboxyl,        sulphonyl, ketone, aldehyde and saturated or unsaturated C₁₋₆        hydrocarbon chain, which C₁₋₆ hydrocarbon chain is substituted        with one or more of nitro, halogen, amino, amido, cyano,        carboxyl, sulphonyl, hydroxyl, ketone, aldehyde or nitrone        groups.

In each aspect of the present invention, the compound of Formula I or Iaor salt thereof may be a compound of Formula II or a salt thereof:

wherein:

-   A) R₁₂ and R₂₆ each independently represent —OH or a glycosidic    functional group; R₁₀, R₁₁, R₁₃ and R₁₄ each independently represent    H, —OH, nitro, halogen, amino, amido, cyano, carboxyl, sulphonyl, a    glycosidic functional group, C₁₋₆ alkoxy-, hydroxyC₁₋₆ alkyl-, C₁₋₆    alkoxy-C₁₋₆ alkyl-, or a saturated or unsaturated C₁₋₆ hydrocarbon    chain which may be substituted with one or more of nitro, halogen,    amino, amido, cyano, carboxyl, sulphonyl, hydroxyl, ketone or    aldehyde groups; and wherein ring B comprises no more than one    glycosidic functional group;-   B) either a):    -   R₂₀ represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon        chain;    -   R₂₁:        -   i) represents H;        -   ii) together with R₂₂ provides a second bond between C¹ and            C²; or        -   iii) when X is —NR₁— and R₁ is not H or C₁₋₆ alkyl, together            with R₁ provides a second bond between C¹ and N;    -   R₂₂:        -   i) represents H;        -   ii) together with R₂₃ forms ═O; or        -   iii) together with R₂, provides a second bond between C¹ and            C²; and    -   R₂₃:        -   i) represents H or a C₂₋₃₀ saturated or unsaturated            hydrocarbon chain; or        -   ii) together with R₂₂ forms ═O;    -   wherein at least one of R₂₀ and R₂₃ is a C₂₋₃₀ saturated or        unsaturated hydrocarbon chain;    -   or b)        -   R₂₀, R₂₁, R₂₂ and R₂₃ form part of a 5, 6 or 7 membered            unsaturated-ring including C¹ and C²(“A” ring), which ring            is substituted with at least one group which is a C₂₋₃₀            saturated or unsaturated hydrocarbon chain, which ring is            optionally and independently further substituted with one or            more groups selected from nitro, halogen, amino, amido,            cyano, carboxyl, sulphonyl, hydroxyl, ketone, aldehyde and            saturated or unsaturated C₂₋₁₅ hydrocarbon chain, which            C₂₋₁₅ hydrocarbon chain may be substituted with one or more            of nitro, halogen, amino, amido, cyano, carboxyl, sulphonyl,            hydroxyl, ketone, aldehyde or nitrone groups;-   C) n is 0 or 1, wherein when n is 1, either i) R₂₄ and R₂₅ together    form ═O, or ii) R₂₄ and R₂₅ represent H;-   D) X is —O—, —S— or wherein R₁ represents i) H or C₁₋₆alkyl, or ii)    together with R₂, provides a second bond between C¹ and N;    wherein said C₂₋₃₀ saturated or unsaturated hydrocarbon chain of    R₂₀, R₂₃ or the 5, 6 or 7 membered unsaturated ring is optionally    and independently substituted with one or more groups selected from    C₁₋₆ alkyl, C₁₋₆alkoxy, hydroxy-C₁₋₆ alkyl, Cl, F, Br, I, —CN,    —CO₂H, —CO₂C₁₋₆alkyl, —S(O)₂C₁₋₆alkyl, —S(O)₂phenyl, —SC₁₋₆alkyl,    —NO₂, —OH, —CF₃, —N(R₂)(R₃), —NHC(O)NHC₁₋₆alkyl, —C(O)N(R₂)(R₃),    imine and substituted or unsubstituted triphenylphosphonium; wherein    one or more available —CH₂— groups present in the C₂₋₃₀ hydrocarbon    chain of R₂₀, R₂₃ or the 5, 6 or 7 membered unsaturated ring is    optionally and independently replaced by —O—, —C(O)—, —S(O)_(p)—, or    —N(R₂)— provided always that no two such substitutions in the    resulting chain are consecutive; wherein R₂ and R₃ each    independently represent H or C₁₋₆alkyl, and wherein p is 0 to 2; and    wherein the total number of ═O on ring C is no greater than 1.

In each aspect of the present invention, X may be O.

In some embodiments of each aspect of the present invention n=0. Inother embodiments of each aspect of the present invention n=1.

In some embodiments of each aspect of the present invention, R₂₀represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon chain;

-   -   R₂₁:    -   i) represents H; or    -   ii) together with R₂₂ provides a second bond between C¹ and C²;    -   R₂₂:    -   i) represents H;    -   ii) together with R₂₃ forms ═O; or    -   iii) together with R₂, provides a second bond between C¹ and C²;        and    -   R₂₃:    -   i) represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon        chain; or    -   ii) together with R₂₂ forms ═O;    -   wherein at least one of R₂₀ and R₂₃ is a C₂₋₃₀ saturated or        unsaturated hydrocarbon chain;

In each aspect of the present invention, R₁₂ and R₂₆ may both representOH or they may both represent a glycosidic functional group; and/or onebut not both of R₁₂ and R₂₆ may represent a glycosidic functional group,for example R₁₂ may be OH when R₂₆ is a glycosidic functional group orvisa versa; and/or one or both of R₁₁ and R₁₃ may represent OH; and/orR₁₀ and R₁₄ each independently represent H, OH or C₁₋₆alkoxy-. Anexample of such a compound, wherein X═O and R₂₇ together with R₂₈provides a second bond between C⁴ and C⁵ is the compound of Formula IIIor a salt thereof:

wherein:A) R₁₀ and R₁₄ each independently represent H, —OH, nitro, halogen,amino, amido, cyano, carboxyl, sulphonyl, a glycosidic functional group,C₁₋₆ alkoxy-, hydroxyC₁₋₆ alkyl-, C₁₋₆ alkoxy-C₁₋₆ alkyl-, or asaturated or unsaturated C₁₋₆ hydrocarbon chain which may be substitutedwith one or more of nitro, halogen, amino, amido, cyano, carboxyl,sulphonyl, hydroxyl, ketone or aldehyde groups; and wherein ring Bcomprises no more than one glycosidic functional group;B) either a):

-   -   R₂₀ represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon        chain;    -   R₂₁:    -   i) represents H; or    -   ii) together with R₂₂ provides a second bond between C¹ and C²;    -   R₂₂:    -   i) represents H;    -   ii) together with R₂₃ forms ═O; or    -   iii) together with R₂, provides a second bond between C¹ and C²;        and    -   R₂₃:    -   i) represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon        chain; or    -   ii) together with R₂₂ forms ═O;    -   wherein at least one of R₂₀ and R₂₃ is a C₂₋₃₀ saturated or        unsaturated hydrocarbon chain;    -   or b)    -   R₂₀, R₂₁, R₂₂ and R₂₃ form part of a 5, 6 or 7 membered        unsaturated-ring including C¹ and C² (“A” ring), which ring is        substituted with a group which is a C₂₋₃₀ saturated or        unsaturated hydrocarbon chain, which ring is optionally and        independently further substituted with one or more groups        selected from nitro, halogen, amino, amido, cyano, carboxyl,        sulphonyl, hydroxyl, ketone, aldehyde and saturated or        unsaturated C₂₋₁₅ hydrocarbon chain, which C₂₋₁₅ hydrocarbon        chain may be substituted with one or more of nitro, halogen,        amino, amido, cyano, carboxyl, sulphonyl, hydroxyl, ketone,        aldehyde or nitrone groups;

-   C) n is 0 or 1, wherein when n is 1, either i) R₂₄ and R₂₅ together    form ═O, or ii) R₂₄ and R₂₅ represent H;    wherein said C₂₋₃₀ saturated or unsaturated hydrocarbon chain of    R₂₀, R₂₃ or the 5, 6 or 7 membered unsaturated ring is optionally    and independently substituted with one or more groups selected from    C₁₋₆ alkyl, C₁₋₆alkoxy, hydroxy-C₁₋₆ alkyl, Cl, F, Br, I, —CN,    —CO₂H, —CO₂C₁₋₆alkyl, —S(O)₂C₁₋₆alkyl, —S(O)₂phenyl, —SC₁₋₆alkyl,    —NO₂, —OH, —CF₃, —N(R₂)(R₃), —NHC(O)NHC₁₋₆alkyl, —C(O)N(R₂)(R₃),    imine and substituted or unsubstituted triphenylphosphonium; wherein    one or more available —CH₂— groups present in the C₂₋₃₀ hydrocarbon    chain of R₂₀, R₂₃ or the 5, 6 or 7 membered unsaturated ring is    optionally and independently replaced by —O—, —C(O)—, —S(O)_(p)—, or    —N(R₂)— provided always that no two such replacements in the    resulting chain are consecutive; wherein R₂ and R₃ each    independently represent H or C₁₋₆alkyl, and wherein p is 0 to 2; and    wherein the total number of ═O on ring C is no greater than 1. A    further example of such a compound, but wherein X═O, n=1, R₂₄    together with R₂₅ forms ═O and R₂₇ together with R₂₈ provides a    second bond between C⁴ and C⁵, is the compound of Formula VI or salt    thereof:

wherein:A) R₁₀ and R₁₄ each independently represent H, —OH, nitro, halogen,amino, amido, cyano, carboxyl, sulphonyl, a glycosidic functional group,C₁₋₆ alkoxy-hydroxy-C₁₋₆ alkyl-, C₁₋₆ alkoxy-C₁₋₆ alkyl-, or a saturatedor unsaturated C₁₋₆ hydrocarbon chain which may be substituted with oneor more of nitro, halogen, amino, amido, cyano, carboxyl, sulphonyl,hydroxyl, ketone or aldehyde groups; and wherein ring B comprises nomore than one glycosidic functional group; and —O-gly represents aglycosidic functional group;B) either a):

-   -   R₂₀ represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon        chain;    -   R₂₁:    -   i) represents H; or    -   ii) together with R₂₂ provides a second bond between C¹ and C²;    -   R₂₂:    -   i) represents H;    -   ii) together with R₂₁ provides a second bond between C¹ and C²;        and    -   R₂₃ represents H, or a C₂₋₃₀ saturated or unsaturated        hydrocarbon chain;    -   wherein at least one of R₂₀ and R₂₃ is a C₂₋₃₀ saturated or        unsaturated hydrocarbon chain;    -   or b)    -   R₂₀, R₂₁, R₂₂ and R₂₃ form part of a 5, 6 or 7 membered        unsaturated-ring including C¹ and C², which ring is substituted        with a group which is a C₂₋₃₀ saturated or unsaturated        hydrocarbon chain, which ring is optionally and independently        further substituted with one or more groups selected from nitro,        halogen, amino, amido, cyano, carboxyl, sulphonyl, hydroxyl,        ketone, aldehyde and saturated or unsaturated C₂₋₁₅ hydrocarbon        chain, which C₂₋₁₅ hydrocarbon chain may be substituted with one        or more of nitro, halogen, amino, amido, cyano, carboxyl,        sulphonyl, hydroxyl, ketone, aldehyde or nitrone groups;        wherein said C₂₋₃₀ saturated or unsaturated hydrocarbon chain of        R₂₀, R₂₃ or the 5, 6 or 7 membered unsaturated ring is        optionally and independently substituted with one or more groups        selected from C₁₋₆ alkyl, C₁₋₆alkoxy, hydroxy-C₁₋₆alkyl, Cl, F,        Br, I, —CN, —CO₂H, —CO₂C₁₋₆alkyl, —S(O)₂C₁₋₆alkyl, —S(O)₂phenyl,        —SC₁₋₆alkyl, —NO₂, —OH, —CF₃, —N(R₂)(R₃), —NHC(O)NHC₁₋₆alkyl,        —C(O)N(R₂)(R₃), imine and substituted or unsubstituted        triphenylphosphonium; wherein one or more available —CH₂— groups        present in the C₂₋₃₀ hydrocarbon chain of R₂₀, R₂₃ or the 5, 6        or 7 membered unsaturated ring is optionally and independently        replaced by —O—, —C(O)—, —S(O)_(p)—, or —N(R₂)— provided always        that no two such replacements in the resulting chain are        consecutive; and wherein R₂ and R₃ each independently represent        H or C₁₋₆alkyl, and wherein p is 0 to 2.

An example of a compound useful in the first aspect of the presentinvention where R₂₀, R₂₁, R₂₂ and R₂₃ form part of a 5, 6 or 7 memberedring is the compound of Formula VII or a salt thereof:

wherein

-   -   R_(A) is a C₂ to C₃₀ saturated or unsaturated hydrocarbon chain;    -   R₁₀, R₁₁, R₁₃, R₁₄ and R₂₆ each independently represent H, OH, a        C₁₋₆ alkoxy, or a saturated or unsaturated C₁₋₆ hydrocarbon        chain which may be substituted with one or more of nitro,        halogen, amino, hydroxyl, ketone or aldehyde group; optionally        there is a double bond between C⁴ and C⁵ of the C ring; and    -   n represents 0 or 1; and    -   R_(B) is a C₂ to C₁₅ saturated or unsaturated hydrocarbon chain,        where when R_(B) is present R_(A) and R_(B) are both C₂ to C₁₂        aliphatic alkyl chains.

The R_(A) group is preferably substituted on ring A at the para positionwith respect to C². The R_(A) group is preferably a C₆₋₁₅ saturated orunsaturated hydrocarbon chain.

Compounds of Formula VII are disclosed in WO 2004/007475 and as such arespecifically excluded from the scope of the second aspect of the presentinvention.

In both aspects of the present invention, the compound of Formula I orIa or salt thereof may be an anthocyanin. Anthocyanins are generallyknown to exist in equilibrium between their hydrated hemiketal form andtheir flavylium cation form, both of which forms are considered to fallwithin the scope of the present invention. Anthocyanins within the scopeof the present invention are compounds of Formula I or salts thereofwherein:

R₁₂ represents OH

R₂₆ represents a glycosidic functional group

R₂₅ together with R₂₇ provide a second bond between C³ and C⁴

R₂₈ represents OH

X═O,

n=1

and R₂₀, R₂₁, R₂₂, and R₂₃ form part of a 6 membered unsaturated-ringincluding C¹ and C², which ring is substituted with at least one groupwhich is a C₂₋₃₀ saturated or unsaturated hydrocarbon chain, which ringis optionally and independently further substituted with one or moregroups selected from nitro, halogen, amino, amido, cyano, carboxyl,sulphonyl, hydroxyl, ketone, aldehyde and saturated or unsaturated C₁₋₆hydrocarbon chain, which C₁₋₆ hydrocarbon chain may be substituted withone or more of nitro, halogen, amino, amido, cyano, carboxyl, sulphonyl,hydroxyl, ketone, aldehyde or nitrone groups. Anthocyanins within thescope of the present invention can be represented by Formula VIIIHH,which is the structural formula of the compound in its hydratedhemiketal form, and Formula VIIIFC, which is the structural formula ofthe compound in its flavylium cation form. The flavylium cation form isalso in equilibrium with the nonionic flavylium form represented byFormula VIIIFH:

In each aspect of the present invention, when R₂₀, R₂₁, R₂₂, and R₂₃ inthe compounds of Formula I or Ia or salts thereof form part of a 5, 6 or7 membered unsaturated-ring including C¹ and C², the ring is substitutedwith a C₂₋₃₀ saturated or unsaturated hydrocarbon chain, as definedabove, at either of the otho, meta or para positions. The ring may beindependently further substituted with one or more groups selected fromnitro, halogen, amino, amido, cyano, carboxyl, sulphonyl, hydroxyl,ketone, aldehyde and saturated or unsaturated C₁₋₆ hydrocarbon chain,which C₁₋₆ hydrocarbon chain may be substituted with one or more ofnitro, halogen, amino, amido, cyano, carboxyl, sulphonyl, hydroxyl,ketone, aldehyde or nitrone groups. By substitution at the orthoposition we mean substitution on a carbon next to the C¹ on the ring. Bysubstitution at the meta position we mean substitution on the carbonnext to the ortho position remote from C₁. By substitution at the paraposition we mean substitution on the carbon next to the meta positionremote from C¹. It will be appreciated by those skilled in the art thatin the case of 5 membered rings, the para position may also be definedas the meta position. In one embodiment, the compound of Formula I orsalt thereof comprises a 5, 6 or 7 membered ring having the C₂₋₃₀hydrocarbon chain substituted at the meta or para position. For example,the compound of Formula I or salt thereof may comprise a 6 membered ringhaving the C₂₋₃₀ hydrocarbon substituted at the meta or para position.

The term “glycosidic functional group” is well known in the art, and isrepresented in the structural formulae herein as —O-gly. For avoidanceof any doubt, however, we mean a carbohydrate group linked to the mainstructure via a glycosidic bond. Preferably, the carbohydrate is asugar. Preferably the sugar is glucose, rhamnose or rutinose.

In each embodiment of the present invention, the C₂₋₃₀ saturated orunsaturated hydrocarbon chain of R₂₀, R₂₃ and the 5, 6 or 7 memberedunsaturated ring may have from two to twenty carbon atoms, preferablyfrom six to fifteen carbon atoms. Suitably the hydrocarbon chain has abackbone having two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen oreighteen consecutive carbon atoms.

The C₂₋₃₀ saturated or unsaturated hydrocarbon chain of R₂₀, R₂₃ and the5, 6 or 7 membered unsaturated ring may include a —CH₂— group connectingto C¹, C² or the 5, 6 or 7 membered ring. This means, for example, thatthe C₂₋₃₀ hydrocarbon chain may not be an alkoxy group, though one ormore carbon atoms within the C₂₋₃₀ hydrocarbon chain may be substitutedwith an alkoxy group. To clarify further, the connection is between acarbon atom on the C₂₋₃₀ saturated or unsaturated hydrocarbon chain andC¹, C² or the 5, 6 or 7 membered ring.

The C₂₋₃₀ saturated or unsaturated hydrocarbon chain of R₂₀, R₂₃ and the5, 6 or 7 membered unsaturated ring may be unsubstituted and ispreferably saturated. The C₂₋₃₀ saturated or unsaturated hydrocarbonchain of R₂₀, R₂₃ and the 5, 6 or 7 membered unsaturated ring ispreferably a straight hydrocarbon chain preferably comprising 6 to 15carbon atoms.

When the C₂₋₃₀ saturated or unsaturated hydrocarbon chain is on a 5, 6or 7 membered unsaturated ring, the ring is optionally and independentlyfurther substituted with one or more groups selected from nitro,halogen, amino, amido, cyano, carboxyl, sulphonyl, ketone, aldehyde andsaturated or unsaturated C₂₋₁₅ hydrocarbon chain, which C₂₋₁₅hydrocarbon chain may be substituted with one or more of nitro, halogen,amino, amido, cyano, carboxyl, sulphonyl, hydroxyl, ketone, aldehyde ornitrone groups. In one embodiment, the ring is unsubstituted except forthe C₂₋₃₀ hydrocarbon chain. In another embodiment, the ring may besubstituted with one or more groups selected from —NH2 and saturated orunsaturated C₂₋₁₅ hydrocarbon chain, which C₂₋₁₅ hydrocarbon chain maybe substituted with one or more of nitro, halogen, amino, amido, cyano,carboxyl, sulphonyl, hydroxyl, ketone, aldehyde or nitrone groups.

Examples of specific compounds or salts thereof which are capable ofpreserving living animal cells include:

The compounds of the present invention are useful for in vitropreservation of living animal cells. It will be appreciated by thoseskilled in the art that in some circumstances preservation may not be apermanent state, but here preservation is intended to include areduction in the rate of deterioration of the viability of individualcells or groups of cells. The expression “in vitro preservation ofliving animal cells” will be well understood by those skilled in the artbut, should any doubt arise, it is intended to mean the in vitropreservation of individual cells or groups of cells, including tissuesand organs. As well as being useful for in vitro research purposes, itwill be appreciated by persons skilled in the art that such in vitropreservation of the cells or groups of cells may be for eventualautologous, isogenic, allogenic or xenogenic transplantation.

For example, the compounds of the invention may be used in the in vitropreservation of viable terminally differentiated cells e.g. red bloodcells and lymphocyte cells such as t-cells and b-cells. Said one or moreterminally differentiated cells can be in the form of individualunipotent stem cells or in the form of tissue i.e. a collection ofinterconnected cells that can perform a similar function in vivo.Tissues suitable for preservation include, but are not limited to,epithelium, connective tissue, muscle tissue and nervous tissue.Furthermore, said one or more fully differentiated cells can form or bepart of an organ i.e. a group of tissues that can perform a specificfunction or group of functions in vivo. Organs suitable for preservationinclude, but are not limited to, heart, lungs, brain, eyes, stomach,spleen, bone, pancreas, kidney, liver, intestines, skin and bladder.

The method of the invention is used to preserve living animal cells in aviable non-terminally differentiated state. The expression “preservationof living animal cells in a viable non-terminally differentiated state”will be well understood by those skilled in the art but, should anydoubt arise, it is intended to include the preservation of livingnon-terminally differentiated animal cells such that the cells retainthe ability to divide and produce at least one non-terminallydifferentiated cell type.

Differentiation is the process by which an undifferentiated cell becomesdifferentiated, which in practice means that the undifferentiated cellbecomes committed to a particular cell lineage and/or loses its originalcapacity for differentiating into particular cell types.

Cells that are non-terminally differentiated give rise to variousdifferent cell types and can alternatively be described as partiallydifferentiated cells. Such cells include the cells of the threeembryonic germ cell layers: endoderm, mesoderm and ectoderm. Cells thatare preserved using the methods of the invention also includeundifferentiated cells, which have the capacity to differentiate into amultitude of cell types. Undifferentiated cells that are preserved usingthe methods of the invention are typically stem cells. Cells that arepreserved using the methods of the invention also includede-differentiated cells, which are produced when a partially orterminally differentiated cell reverts to an earlier developmentalstage. De-differentiated cells can therefore be undifferentiated orpartially differentiated. In this aspect, the methods of the inventionare particularly useful for the preservation of de-differentiatedneuronal cells. In addition, the methods of the invention are useful forthe preservation of cells in cell culture which have de-differentiated.

Cells that are non-terminally differentiated include cells that arearrested at a particular stage of the differentiation process or thede-differentiation process. Cells that are non-terminally differentiatedtherefore include cells that are arrested at a particular stage whilstbecoming committed to a particular cell lineage, cells that are arrestedat a particular stage whilst reverting to an earlier developmental stageafter being non-terminally or terminally differentiated and cells thatare re-differentiated after becoming de-differentiated.

In one embodiment, the present invention finds use in the preservationof cells that are differentiated to a non-terminal state ofdifferentiation, i.e. partially differentiated cells, before beingtransported or stored. The cells can then be induced to a terminallydifferentiated state when required.

In this aspect of the invention, the cells that are preserved by themethods of the invention can be used for transplantation. Typically,such cells are partially differentiated, and retain the capacity todifferentiate into a number of different cell types.

Undifferentiated animal cells which can be preserved using the methodsof the invention are typically stem cells. Stem cells are unspecialisedcells which are capable of differentiating into various different typesof cells and which are capable of self-renewal. Stem cells which can bepreserved using a method of the present invention include totipotentstem cells (capable of differentiating into embryonic and extraembryoniccell types), pluripotent stem cells (capable of differentiating intoendoderm, mesoderm and ectoderm germ layers), and multipotent stem cells(capable of differentiating into a plurality of closely related cells).

Types of stem cells which can be preserved using the methods of thepresent invention include embryonic stem (ES) cells (ESCs), adult stemcells and induced pluripotent stem (iPS) cells. Embryonic stem cells arederived from the blastocyst of a mammalian embryo and are totipotent.Embryonic stem cells were originally described by Evans and Kaufman(Nature, 292(5819): 154-156, 1981). Adult stem cells are pluripotent,and include hematopoietic stem cells and mesenchymal stem cells. Inducedpluripotent cells are artificially derived from a non-pluripotent cellsuch as an adult somatic cell by the insertion of certain genes and arevery similar to embryonic stem cells (Takahashi et al, Cell 131(5):861-872, 2007; and Yu et al, Science 318(5858), 1917-1920, 2007). Stemcells are also found in the blood of the umbilical cord, and suchumbilical cord blood stem cells can also be preserved using the methodsof the present invention.

Stem cells which can be preserved using the methods of present inventioncan be human or non-human. Typically, the stem cell is a mouse embryonicstem cell or a human embryonic stem cell. Stem cells which can bepreserved using the methods of present invention include transgenic stemcells. Stem cells which can be preserved using the methods of presentinvention also include those produced from hybrid embryos or cytoplasmichybrid (cybrid) embryos.

Other types of undifferentiated cells which can be preserved using themethods of the present invention include embryonic germ (EG) cells andembryonic carcinoma (EC) cells.

Undifferentiated cells can be identified, for example, by expression ofparticular marker genes. For example, stem cells can be identified bythe expression of the marker genes Oct3/4 and Nanog. Expression of suchmarker genes can be determined by any suitable method known in the art,for example VCR.

The undifferentiated animal cells which can be preserved using a methodof the present invention are typically mammalian cells. Such mammaliancells include human and non-human cells. For example, non-human cellscan be from rodents, such as mice, rats and guinea pigs; ungulates, suchas cattle, sheep, goats and pigs; or other mammals such as cats, dogs,horses or rabbits. The animal cells can alternatively be bird cells, forexample from chickens or turkeys, or fish cells, for example from zebrafish or salmon.

The compounds of Formula I or salts thereof may be used for preservingliving animal cells by any of the relevant methods disclosed in EuropeanPatent Publication No. EP-A-1057405, contents of which are hereinincorporated by in its entirety.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

To the extent not already indicated, it will be understood by those ofordinary skill in the art that any one of the various embodiments hereindescribed and illustrated may be further modified to incorporatefeatures shown in any of the other embodiments disclosed herein.

The following examples will demonstrate some embodiments of theinvention. The examples are illustrative, and are not intended to limit,in any manner, the claimed invention.

EXAMPLES Representative Compound Synthesis

Compounds of Formula I and Ia wherein R₂₀, R₂₁, R₂₂, and R₂₃ form partof a 6 membered unsaturated ring including C¹ and C² may be formed bythe process described in WO 2004/007475, the contents of which areherein incorporated by reference in its entirety. A person skilled inthe art would readily be able to adapt this process for the manufactureof compounds of Formula I or I a including a 5 or 7 membered ring.

Compounds of Formula I or Ia wherein R₂₀, R₂₁, R₂₂, and R₂₃ do not formpart of a 6 membered unsaturated ring including C¹ and C² may be formedby the following process.

The use of an in situ quench protocol gave the desired boronic acidwhich would eventually be used to form the B-ring of the target compound(Scheme 1).

Phosphonate ester (11) was prepared as follows:

To a stirred solution of 5 (1.88 g, 8.52 mmol, 1.00 eq.) in THF (50 mL)was added benzyl alcohol (4.40 mL, 4.60 g, 42.6 mmol, 5.00 eq.), thentriphenylphosphine (2.23 g, 8.52 mmol, 1.00 eq.). The reaction wascooled to 0° C. and DIAD (1.7 mL, 1.72 g, 8.52 mmol, 1.00 eq.) was addeddropwise. The reaction was warmed to room temperature and stirred for 4hours. The reaction mixture was concentrated in vacuo and purified byflash chromatography (10% EtOAc:90% petrol→20% EtOAc:80% petrol→30%EtOAc:70% petrol→50% EtOAc:50% petrol) to yield the benzylated compoundas a yellow oil containing about 30% reduced DIAD (1.01 g, 38% includingimpurity). The crude benzylated compound (535 mg, 1.72 mmol, 1.00 eq.)was diluted with CH₂Cl₂ (5 mL) and SOCl2 (188 μL, 2.58 mmol, 1.50 eq.)was added at 0° C. The reaction was stirred, with warming to roomtemperature, for 2.5 hours, when complete by HPLC. The reaction mixturewas concentrated in vacuo and purified by flash chromatography (10%EtOAc:90% petrol→20% EtOAc:80% petrol) to yield a brown oil (276 mg,49%). The chloride (261 mg, 0.79 mmol, 1.00 eq.) was taken intotrimethylphosphite (2 mL) and the reaction was heated to reflux andstirred for 16 hours. The reaction was cooled to room temperature andconcentrated in vacuo. Purification by flash chromatography (70%EtOAc:30% petrol→100% EtOAc) gave the title phosphonate ester 11 as abrown oil (233 mg, 73%) ¹H-NMR (400 MHz, CDCl₃) 7.47-7.43 (2H, m,2×ArH), 7.38 7.31 (3H, m, 3×ArH), 6.35 (1H, d, J 3.0, vinylic H), 5.25(2H, s, OCH2Ar), 3.80 (6H, d, J 11.1, 2×OCH3), 3.07 (2H, d, J 21.7,CH2P).

To 11 (400 mg, 0.99 mmol, 1.00 eq.) in 1:1 DMF/THF (8 mL) at −78° C. wasadded LHMDS (1.0 M in THF; 1.10 mL, 1.09 mmol, 1.10 eq.) and thereaction was stirred at that temperature for 10 mins (yellow/orangecolour). Heptaldehyde (113 mg, 0.99 mmol, 1.00 eq.) in DMF (4 mL) wasadded in one portion at −78° C. and the reaction was stirred at thattemperature for 45 mins. The reaction was warmed to 0° C. and stirredfor 10 mins until complete by HPLC. The reaction was quenched withsaturated aqueous ammonium chloride solution (5 mL) and extracted intoEtOAc (3×10 mL). The combined organic extracts were washed with water(10 mL) and brine (10 mL), dried (MgSO4) and concentrated in vacuo.Purification by flash chromatography (10% EtOAc:90% petrol) gave thealkene 12b as a colourless oil and a 2.5:1.0 mixture of E and Z isomersrespectively (251 mg, 65%) ¹H-NMR (400 MHz, CDCl₃) 7.50-7.45 (2H, m,2×ArH), 7.40-7.30 (3H, m, 3×ArH), 6.62 (0.7H, dt, J 15.7 and 7.1,vinylic H), 6.24 (0.3H, s, HC═CO); 6.16 (0.7H, s, HC═CO), 6.08-5.98(0.3H, m, vinylic H), 6.01 (0.7H, dt, J 15.7 and 1.5, vinylic H), 5.92(0.3H, dt, J 12.1 and 1.52, vinylic H), 5.28 (0.6H, s, OCH2Ar), 5.26(1.5H, s, OCH2Ar), 2.46 (0.6H, ddd, J 13.1, 7.5 and 1.5, allylic CH2),2.24 (1.5H, ddd, J 13.1, 7.5 and 1.5, allylic CH2), 1.52-1.44 (2H, m,CH2), 1.40-1.24 (6H, m, CH2), 0.93-0.88 (3H, m, CH3).

To 11 (400 mg, 0.99 mmol, 1.00 eq.) in 1:1 DMF/THF (8 mL) at −78° C. wasadded LHMDS (1.0 M in THF; 1.10 mL, 1.09 mmol, 1.10 eq.) and thereaction was stirred at that temperature for 10 mins (yellow/orangecolour). Undecanal (168 mg, 0.99 mmol, 1.00 eq.) in DMF (4 mL) was addedin one portion at −78° C. and the reaction was stirred at thattemperature for 45 mins. The reaction was warmed to 0° C. and stirredfor 10 mins until complete by HPLC. The reaction was quenched withsaturated aqueous ammonium chloride solution (5 mL) and extracted intoEtOAc (3×10 mL). The combined organic extracts were washed with water(10 mL) and brine (10 mL), dried (MgSO4) and concentrated in vacuo.Purification by flash chromatography (5% EtOAc:95% petrol→10%EtOAc:petrol) gave the alkene 12c as a colourless oil and an approximate3:1 mixture of E and Z isomers respectively (336 mg, 76%) ¹H-NMR (400MHz, CDCl₃) 7.50-7.45 (2H, m, 2×ArH), 7.40-7.30 (3H, m, 3×ArH), 6.61(0.7H, dt, J 15.7 and 7.1, vinylic H), 6.24 (0.3H, s, HC═CO), 6.16(0.7H, s, HC═CO), 6.07-5.99 (0.3H, m, vinylic H), 6.00 (0.7H, dt, J 16.1and 1.5, vinylic H), 5.92 (0.3H, dt, J 11.6 and 1.5, vinylic H), 5.28(0.6H, s, OCH2Ar), 5.25 (1.4H, s, OCH2Ar), 2.46 (0.5H, ddd, J 15.2, 7.6and 1.5, allylic H), 2.24 (1.5H, ddd, J 15.2, 7.6 and 1.5, allylic H),1.52-1.42 (2H, m, CH2), 1.40-1.22 (14H, m, 7×CH2), 0.90 (3H, t, J 7.1,CH3).

A suspension of 12b (251 mg, 0.64 mmol, 1.00 eq.), Pd(PPh3)4 (69 mg,0.06 mmol, 10 mol %) and K2CO3 (283 mg, 2.05 mmol, 3.20 eq.) in DMF/H2O(2:1, 5.1 mL) was degassed for 5 mins, then a degassed solution ofboronic acid (310 mg, 0.71 mmol, 1.10 eq.) in DMF (1.8 mL) and thereaction was heated to 60° C. and stirred for 4 hours. The reaction wascooled to room temperature, diluted with water (20 mL) and extractedwith EtOAc (2×20 mL). The combined organic extracts were dried (MgSO4)and concentrated in vacuo. Purification by flash chromatography (10%EtOAc:90% petrol→20% EtOAc:80% petrol→30% EtOAc:70% petrol→40% EtOAc:60%petrol) gave the title compound as a yellow oil (332 mg, 74%) ¹H-NMR(400 MHz, CDCl₃) 7.48-7.20 (22H, m, 22×ArH), 6.52-5.90 (3H, m, vinylic Hand pyranone H), 5.32-4.92 (8H, m, 4×OCH2Ar), 2.60-2.20 (2H, m, CH2),1.58-1.40 (2H, m, CH2), 1.40-1.20 (6H, m, 3×CH2), 1.00-0.80 (3H, m,CH3).

A suspension of 12c (204 mg, 0.46 mmol, 1.00 eq.), Pd(PPh3)4 (53 mg,0.05 mmol, 10 mol %) and K2CO3 (203 mg, 1.47 mmol, 3.20 eq.) in DMF/H2O(2:1, 5.1 mL) was degassed for 5 mins, then a degassed solution ofboronic acid 221 mg, 0.50 mmol, 1.10 eq.) in DMF (1.8 mL) and thereaction was heated to 60° C. and stirred for 4 hours. The reaction wascooled to room temperature, diluted with water (20 mL) and extractedwith EtOAc (2×20 mL). The combined organic extracts were dried (MgSO4)and concentrated in vacuo. Purification by flash chromatography (10%EtOAc:90% petrol→20% EtOAc:80% petrol→>30% EtOAc:70% petrol→40%EtOAc:60% petrol) gave the title compound as a yellow oil (260 mg, 74%)¹H-NMR (400 MHz, CDCl₃) 7.50-7.20 (22H, m, 22×ArH), 6.50-6.00 (3H, m,3×vinylic H), 5.30-4.90 (6H, m, OCH2Ar), 2.56-2.48 (0.4H, m, CH2),2.28-2.20 (1.4H, m, CH2), 1.57-1.40 (2H, m, CH2), 1.40-1.20 (14H, m,7×CH2), 0.92-0.84 (3H, m, CH3).

13b (320 mg, 0.45 mmol, 1.00 eq.) was dissolved in the minimal amount ofethyl acetate and taken into MeOH (10 mL). 10% Pd/C (180 mg) wasslurried with MeOH (1 mL) and added to the solution. The solution wasevacuated and backfilled with H2 3 times and stirred for 1 hour. Thesuspension was filtered through Celite® and concentrated in vacuo toyield 14b as a tan solid (105 mg, 67%) ¹H-NMR (400 MHz, d⁶-DMSO/D2Oshake) 7.06 (2H, s, 2×ArH), 6.24 (1H, s, vinylic H), 2.59 (2H, t, J 7.1,CH2), 1.64 (2H, quintet, J 7.1, CH2), 1.30-1.10 (10H, m, 5×CH2), 0.81(3H, t, J 7.1, CH3).

13c (250 mg, 0.33 mmol, 1.00 eq.) was dissolved in the minimal amount ofethyl acetate and taken into MeOH (10 mL). 10% Pd/C (150 mg) wasslurried with MeOH (1 mL) and added to the solution. The solution wasevacuated and backfilled with H2 3 times and stirred for 1 hour. Thesuspension was filtered through Celite® and concentrated in vacuo toyield 14c as a tan solid (84 mg, 63%) ¹H-NMR (400 MHz, d⁶-DMSO/D2Oshake) 7.08 (2H, s, 2×ArH), 6.24 (1H, s, vinylic H), 2.59 (2H, t, J 7.1,CH2), 1.64 (2H, quintet, J 7.1, CH2), 1.37-1.00 (18H, m, 9×CH2), 0.81(3H, t, J 7.1, CH3).

EXPERIMENTAL

The following experiments were conducted to demonstrate the protectiveeffective of certain antioxidant compounds in relation to oxidativestress-induced cytotoxicity in mouse embryonic stem cells:

Oxidative stress and related free radical damage can be induced incells, including stem cells, by exposing the cells to tert-butylhydroperoxide in the incubation medium. Decomposition of the peroxideover a period of time leads to a steady generation of reactive oxygenspecies. This in turn triggers a cascade of free radical-mediated eventsthat eventually leads to irreversible damage and ultimately cell deathas the natural antioxidant defences are overwhelmed.

The extent of cytotoxicity induced by peroxide exposure can bequantified using a number of well-documented assays, including the MTTprotocol. This is a colorometric assay that measures the reduction ofMTT to formazan, a process that can only occur when cells are viable.Thus, by comparing the amount of formazan produced in control cells,against cells treated with a cytotoxic agent, such as tert-butylhydroperoxide, the reduction in cell viability induced by the agent canbe measured.

The assay can be further extended to determine the effectiveness ofcompounds to provide protection against cytotoxic agents. In the case ofthe novel antioxidants, the subject of this patent, this can be achievedby pre-incubation of the cells with the antioxidant agent prior toinduction of oxidative stress by tert-butyl hydroperoxide. Thus acomparison of the amount of formazan produced by control cells againstcells with the cytotoxic peroxide present, plus or minus pre-incubationwith the novel antioxidant, can determine the extent by which thecompound can retain cell viability.

(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

A 96-well plate was seeded with approximately 20,000 mouse embryonicstem cells per well and left to grow at 37° C. (5% CO₂) overnight.Antioxidants, denoted as Exp.1, Exp.2 and Exp.3, were dissolved in DMSOand added to the wells in triplicate 30 min before induction ofoxidative stress with tert-butyl hydroperoxide. The resultantconcentrations of the tested antioxidants in the wells were 0.1, 0.5 and1 μM. Controls were also initiated using cell medium (DMEM⁺)/DMSO. Afterthe 30 min pre-incubation period, tert-butyl peroxide was added to thewells to provide a final concentration of 400 μM. The plated cells werereturned to the incubator (37° C.) for 90 min. After this period, 20 μLof the MTT reagent solution was added to all of the wells. The plate wasthen further incubated for a further 3 hours. The supernatant wasremoved from the wells and 200 μl DMSO added to all wells to solubilisereduced MTT (formazan). The plate was protected from light and shakenfor 10 min. The supernatant was removed from the wells and aliquotedinto a new 96-well plate for the colorometric measurements. Absorbancewas determined at 540 nm.

RESULTS AND DISCUSSION

The average corrected absorbance. (0.737) for the DMEM⁺ control (whichcontained no antioxidant compounds or tert-butyl hydroperoxide)represented the 100% viability benchmark. Comparison of the correctedabsorbances in the various well treatment regimes relative to that ofthe DMEM⁺ control enabled the resultant effects on cell viability to becalculated. tBHP=tert-butyl hydroperoxide.

From Table 1, exposure to tert-butyl hydroperoxide reduces cellviability to 37% of the control. At a concentration of 1 μM in theincubation medium without the peroxide present, neither Exp.1 nor Exp.2had a negative impact on viability indicating that neither compoundinduced cytotoxicity at that level of exposure.

Exp.1, Exp.2 and Exp.3 all exhibited a strong protective effect from theoxidative stress-induced cytotoxicity of tert-butyl hydroperoxide. Exp.2was of particular note in this respect and was able to almost fullyreverse the loss of cell viability induced by tert-butyl hydroperoxide(96.9%+/−3.9 sd). A concentration dependence effect was observed.

In respect of structure activity relationships, it is evident that thedodecyl chain of Exp.2 imparts greater efficacy than the octyl chain ofExp.3. A key finding of these results is in relation to the literatureon natural flavonoids in which a number of authors have alluded to theimportance of the A-ring of the flavonoid as being an integral componentof the antioxidant activity of the molecule (as exemplified in“Structure-Antioxidant Activity Relationships Of Flavonoids And PhenolicAcids; Rice-Evans et al.; Free Radical Biology & Medicine, Vol. 20, No.7, pp 933-956, 1996”)

The data presented here unequivocally demonstrates that the A-ringconstituent is not pre-requisite and can be completely removed from themolecule with retention of potent antioxidant bio-activity. Comparisonof Exp.1 relative to Exp.2 in this cell viability oxidative stress assayindicates that the activity may not just be preserved by removal of theA-ring, but enhanced.

TABLE 1 Ave. Ave. Corrected Std Dev. Ave. Cell Std Dev. TreatmentsAbsorbance Absorbance (±) Viability (%) (±) Controls DMEM⁺ 0.811 0.7370.027 100.0 3.6 400 μM tBHP 0.347 0.273 0.010 37.0 1.3 1 μM Exp.1 0.8660.791 0.015 107.4 2.0 (No tBHP) 1 μM Exp.2 0.841 0.767 0.044 104.1 5.9(No tBHP) Exp. 1 (μM) 400 μM tBHP 0.1 0.494 0.420 0.042 56.9 5.7 0.50.712 0.638 0.041 86.5 5.5 1   0.700 0.625 0.026 84.8 3.5 Exp. 2 (μM)400 μM tBHP 0.1 0.520 0.446 0.013 60.5 1.7 0.5 0.773 0.699 0.047 94.86.4 1   0.788 0.714 0.029 96.9 3.9 Exp. 3 (μM) 400 μM tBHP 0.1 0.3280.254 0.020 34.4 2.7 0.5 0.483 0.409 0.031 55.5 4.2 1   0.593 0.5190.026 70.4 3.5 Compounds Tested Exp.1:

Exp.2:

Exp.3:

1. A method of in vitro preservation of living animal cells in a viablenon-terminally differentiated state, said method comprising contactingliving non-terminally differentiated animal cells with a compound ofFormula I or a salt thereof:

wherein: A) R₁₂ and R₂₆ each independently represent —OH or a glycosidicfunctional group; R₁₀, R₁₁, R₁₃, and R₁₄ each independently represent H,—OH, nitro, halogen, amino, amido, cyano, carboxyl, sulphonyl, aglycosidic functional group, C₁₋₆ alkoxy-, hydroxy-C₁₋₆ alkyl-, C₁₋₆alkoxy-C₁₋₆ alkyl-, or a saturated or unsaturated C₁₋₆ hydrocarbon chainwhich may be substituted with one or more of nitro, halogen, amino,amido, cyano, carboxyl, sulphonyl, hydroxyl, ketone or aldehyde groups;and wherein ring B comprises no more than one glycosidic functionalgroup; B) either a): R₂₀ represents H or a C₂₋₃₀ saturated orunsaturated hydrocarbon chain; R₂₁: i) represents H; ii) together withR₂₂ provides a second bond between C¹ and C²; or iii) when X is —NR₁—and R₁ is not H or C₁₋₆alkyl, together with R₁ provides a second bondbetween C¹ and N; R₂₂: i) represents H; ii) together with R₂₃ forms ═O;or iii) together with R₂₁provides a second bond between C¹ and C²; R₂₃:i) represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon chain;ii) together with R₂₂ forms ═O; wherein at least one of R₂₀ and R₂₃ is aC₂₋₃₀ saturated or unsaturated hydrocarbon chain; or b): R₂₀, R₂₁, R₂₂,and R₂₃, together with C¹ and C², form a 5, 6 or 7 memberedunsaturated-ring, which ring is substituted with a group which is aC₂₋₃₀ saturated or unsaturated hydrocarbon chain, which ring isoptionally and independently further substituted with one or more groupsselected from nitro, halogen, amino, amido, cyano, carboxyl, sulphonyl,hydroxyl, ketone, aldehyde and saturated or unsaturated C₂₋₁₅hydrocarbon chain, which C₂₋₁₅ hydrocarbon chain may be substituted withone or more of nitro, halogen, amino, amido, cyano, carboxyl, sulphonyl,hydroxyl, ketone, aldehyde or nitrone groups; C) n is 0 or 1, whereinwhen n is 0, either i) R₂₇ and R₂₈ represent H or ii) R₂₇ together withR₂₈ provide a second bond between C⁴ and C⁵; or when n is 1, either i)R₂₄ and R₂₅ together form ═O and R₂₇ and R₂₈ represent H or R₂₇ togetherwith R₂₈ provide a second bond between C⁴ and C⁵, or ii) R₂₄ and R₂₅represent H and R₂₇ and R₂₈ represent H or R₂₇ together with R₂₈ providea second bond between C⁴ and C⁵ or iii) R₂₄ represents H, R₂₅ togetherwith R₂₇ provide a second bond between C³ and C⁴, R₂₈ represents —OH andX is —O—; D) X is —O—, —S— or —NR₁—, wherein R₁ i) represents H orC₁₋₆alkyl, or ii) together with R₂₁ provides a second bond between C¹and N; wherein said C₂₋₃₀ saturated or unsaturated hydrocarbon chain ofR₂₀, R₂₃ and the 5, 6 or 7 membered unsaturated ring is optionally andindependently substituted with one or more groups selected from C₁₋₆alkyl, C₁₋₆alkoxy, hydroxy-C₁₋₆ alkyl, Cl, F, Br, I, —CN, —CO₂H,—CO₂C₁₋₆alkyl, —S(O)₂C₁₋₆alkyl, —S(O)₂phenyl, —SC₁₋₆alkyl, —NO₂, —OH,—CF3, —N(R₂)(R₃), —NHC(O)NHC₁₋₆alkyl, —C(O)N(R₂)(R₃), imine andsubstituted or unsubstituted triphenylphosphonium; and wherein one ormore available —CH₂-groups present in the C₂₋₃₀ hydrocarbon chain ofR₂₀, R₂₃ and the 5, 6 or 7 membered unsaturated ring is optionally andindependently replaced by —O—, —C(O)—, —S(O)_(p)—, or —N(R₂)— providedalways that no two such replacements in the resulting chain areconsecutive; wherein R₂ and R₃ each independently represent H orC₁₋₆alkyl, and wherein p is 0 to 2; and wherein the total number of ═Oon ring C is no greater than
 1. 2. The method of claim 1, wherein Xrepresents —O—.
 3. The method of claim 1, wherein R₁₂ and R₂₆ bothrepresent —OH.
 4. The method of claim 1, wherein one of R₁₂ and R₂₆represents —OH and the other of R₁₂ and R₂₆ represents a glycosidicfunctional group.
 5. The method of claim 1, wherein n=1.
 6. The methodof claim 1, wherein n=0.
 7. The method of claim 1, wherein: R₂₀represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon chain; R₂₁:i) represents H; or ii) together with R₂₂ provides a second bond betweenC¹ and C²; R₂₂: i) represents H; ii) together with R₂₃ forms ═O; or iii)together with R₂, provides a second bond between C¹ and C²; and R₂₃: i)represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon chain; orii) together with R₂₂ forms ═O.
 8. The method of claim 1, wherein R₂₀,R₂₁, R₂₂ and R₂₃, together with C¹ and C², form a 5, 6 or 7 memberedunsaturated-ring.
 9. The method of claim 8, wherein saidunsaturated-ring is substituted with a C₂₋₃₀ saturated or unsaturatedhydrocarbon chain at the meta or para position relative to C¹ or whereinsaid unsaturated ring is substituted with C₂₋₁₅ saturated or unsaturatedhydrocarbon chains at two of the ortho, meta and para positions relativeto C¹.
 10. The method of claim 1, wherein said C₂₋₃₀ saturated orunsaturated hydrocarbon chain of R₂₀, R₂₃ and the 5, 6 or 7 memberedunsaturated ring comprises a —CH₂-group connecting to C¹, C² or the 5, 6or 7 membered ring.
 11. The method of claim 10, wherein said C₂₋₃₀saturated or unsaturated hydrocarbon chain of R₂₀, R₂₃ and the 5, 6 or 7membered unsaturated ring is unsubstituted.
 12. The method of claim 11,wherein said C₂₋₃₀ saturated or unsaturated hydrocarbon chain of R₂₀,R₂₃ and the 5, 6 or 7 membered unsaturated ring is saturated.
 13. Themethod of claim 10, wherein said C₂₋₃₀ saturated or unsaturatedhydrocarbon chain of R₂₀, R₂₃ and the 5, 6 or 7 membered unsaturatedring is a straight hydrocarbon chain comprising 6 to 15 carbon atoms.14. The method of claim 1, wherein the living animal cells are stemcells.
 15. The method of claim 14, wherein the stem cells are selectedfrom the group consisting of embryonic stem cells (ESCs), adult stemcells and induced pluripotent stem (iPS) cells.
 16. The method of claim15, wherein the embryonic stem cells are human embryonic stem cells ormouse embryonic stem cells.
 17. The method of claim 15, wherein the stemcells are induced pluripotent stem (iPS) cells.
 18. The method of claim1, wherein the living animal cells are de-differentiated cells.
 19. Acompound of Formula Ia or a salt thereof:

wherein: i. R₁₂ and R₂₆ each independently represent —OH or a glycosidicfunctional group; R₁₀, R₁₁, R₁₃, and R₁₄ each independently represent H,—OH, nitro, halogen, amino, amido, cyano, carboxyl, sulphonyl, aglycosidic functional group, C₁₋₆ alkoxy-, hydroxy-C₁₋₆ alkyl-, C₁₋₆alkoxy-C₁₋₆ alkyl-, or a saturated or unsaturated C₁₋₆ hydrocarbon chainwhich may be substituted with one or more of nitro, halogen, amino,amido, cyano, carboxyl, sulphonyl, hydroxyl, ketone or aldehyde groups;and wherein ring B comprises no more than one glycosidic functionalgroup; ii. either a): R₂₀ represents H or a C₂₋₃₀ saturated orunsaturated hydrocarbon chain; R₂₁: i) represents H; ii) together withR₂₂ provides a second bond between C¹ and C²; or iii) when X is —NR₁—and R₁ is not H or C₁₋₆alkyl, together with R₁ provides a second bondbetween C¹ and N; R₂₂: i) represents H; ii) together with R₂₃ forms ═O;or iii) together with R₂, provides a second bond between C¹ and C²; R₂₃:i) represents H or a C₂₋₃₀ saturated or unsaturated hydrocarbon chain;ii) together with R₂₂ forms ═O; wherein at least one of R₂₀ and R₂₃ is aC₂₋₃₀ saturated or unsaturated hydrocarbon chain; or b): R₂₀, R₂₁, R₂₂,and R₂₃, together with C¹ and C², form a 5, 6 or 7 memberedunsaturated-ring, which ring is substituted with a group which is aC₂₋₃₀ saturated or unsaturated hydrocarbon chain, which ring isoptionally and independently further substituted with one or more groupsselected from nitro, halogen, amino, amido, cyano, carboxyl, sulphonyl,ketone, aldehyde and saturated or unsaturated C₂₋₁₅ hydrocarbon chain,which C₂₋₁₅hydrocarbon chain may be substituted with one or more ofnitro, halogen, amino, amido, cyano, carboxyl, sulphonyl, hydroxyl,ketone, aldehyde or nitrone groups; C) n is 0 or 1, wherein when n is 0,either i) R₂₇ and R₂₈ represent H or ii) R₂₇ together with R₂₈ provide asecond bond between C⁴ and C⁵; or when n is 1, either i) R₂₄ and R₂₅together form ═O and R₂₇ and R₂₈ represent H or R₂₇ together with R₂₈provide a second bond between C⁴ and C⁵, or ii) R₂₄ and R₂₅ represent Hand R₂₇ and R₂₈ represent H or R₂₇ together with R₂₈ provide a secondbond between C⁴ and C⁵ or iii) R₂₄ represents H, R₂₅ together with R₂₇provide a second bond between C³ and C⁴, R₂₈ represents —OH and X is—O—; D) X is —O—, —S— or —NR₁—, wherein R₁ i) represents H or C₁₋₆alkyl,or ii) together with R₂, provides a second bond between C¹ and N;wherein said C₂₋₃₀ saturated or unsaturated hydrocarbon chain of R₂₀,R₂₃ and the 5, 6 or 7 membered unsaturated ring is optionally andindependently substituted with one or more groups selected from C₁₋₆alkyl, C₁₋₆alkoxy, hydroxy-C₁₋₆ alkyl, Cl, F, Br, I, —CN, —CO₂H,—CO₂C₁₋₆alkyl, —S(O)₂C₁₋₆alkyl, —S(O)₂-phenyl, —SC₁₋₆alkyl, —NO₂, —OH,—CF3, —N(R₂)(R₃), —NHC(O)NHC₁₋₆alkyl, —C(O)N(R₂)(R₃), imine andsubstituted or unsubstituted triphenylphosphonium; and wherein one ormore available —CH₂— groups present in the C₂₋₃₀ hydrocarbon chain ofR₂₀, R₂₃ and the 5, 6 or 7 membered unsaturated ring is optionally andindependently replaced by —O—, —C(O)—, —S(O)_(p)—, or —N(R₂)— providedalways that the resulting chain includes a —CH₂— group connecting to C¹,C² or the 5, 6 or 7 membered ring and no two such replacements areconsecutive; wherein R₂ and R₃ each independently represent H orC₁₋₆alkyl, and wherein p is 0 to 2; and wherein the total number of ═Oon ring C is no greater than 1; provided that when i) n=1, ii) Xrepresents —O—, iii) R₁₂ represents —OH, iv) R₂₄ together with R₂₅represent ═O, v) R₂₀, R₂₁, R₂₂ and R₂₃ form a benzene ring including C¹and C², and vi) said benzene ring is substituted with at least one groupwhich is a C₂₋₃₀ saturated or unsaturated hydrocarbon chain, then: saidC₂₋₃₀ saturated or unsaturated hydrocarbon chain is substituted with oneor more groups selected from C₁₋₆alkoxy, hydroxy-C₁₋₆ alkyl, Cl, F, Br,I, —CN, —CO₂H, sulphonyl, —CO₂C₁₋₆alkyl, —S(O)₂C₁₋₆alkyl, —S(O)₂phenyl,—SC₁₋₆alkyl, —NO₂, —OH, —CF₃, —N(R₂)(R₃), —NHC(O)NHC₁₋₆alkyl,—C(O)N(R₂)(R₃), imine and substituted or unsubstitutedtriphenylphosphonium; and/or wherein one or more available —CH₂— groupspresent in said C₂₋₃₀ hydrocarbon chain is replaced by —O—, —C(O)—,—S(O)_(p)—, or —N(R₂)—; wherein R₂ and R₃ each independently represent Hor C₁₋₆alkyl, and wherein p is 0 to 2; and/or said benzene ring issubstituted with one or more groups selected from nitro, halogen, amino,amido, cyano, carboxyl, sulphonyl, ketone, aldehyde and saturated orunsaturated C₁₋₆ hydrocarbon chain, which C₁₋₆ hydrocarbon chain issubstituted with one or more of nitro, halogen, amino, amido, cyano,carboxyl, sulphonyl, hydroxyl, ketone, aldehyde or nitrone groups. 20.The compound of claim 19, wherein X represents —O—.
 21. The compound ofclaim 19, wherein R₁₂ and R₂₆ both represent —OH.
 22. The compound ofclaim 19, wherein one of R₁₂ and R₂₆ represents —OH and the other of R₁₂and R₂₆ represents a glycosidic functional group.
 23. The compound ofclaim 19, wherein n=1.
 24. The compound of claim 19, wherein n=0. 25.The compound of claim 19, wherein: R₂₀ represents H or a C₂₋₃₀ saturatedor unsaturated hydrocarbon chain; R₂₁: i) represents H; or ii) togetherwith R₂₂ provides a second bond between C¹ and C²; R₂₂: i) represents H;ii) together with R₂₃ forms ═O; or iii) together with R₂₁ provides asecond bond between C¹ and C²; and R₂₃: i) represents H or a C₂₋₃₀saturated or unsaturated hydrocarbon chain; or ii) together with R₂₂forms ═O.
 26. The compound of claim 19, wherein R₂₀, R₂₁, R₂₂ and R₂₃,together with C¹ and C², form a 5, 6 or 7 membered unsaturated-ring. 27.The compound of claim 26, wherein said unsaturated-ring is substitutedwith a C₂₋₃₀ saturated or unsaturated hydrocarbon chain at the meta orpara position relative to C¹ or wherein said unsaturated ring issubstituted with C₂₋₁₅ saturated or unsaturated hydrocarbon chains attwo of the ortho, meta and para positions relative to C¹.
 28. Thecompound of claim 19, wherein said C₂₋₃₀ saturated or unsaturatedhydrocarbon chain of R₂₀, R₂₃ and the 5, 6 or 7 membered unsaturatedring comprises a —CH₂— group connecting to C¹, C² or the 5, 6 or 7membered ring.
 29. The compound of claim 28, wherein said C₂₋₃₀saturated or unsaturated hydrocarbon chain of R₂₀, R₂₃ and the 5, 6 or 7membered unsaturated ring is unsubstituted.
 30. The compound of claim29, wherein said C₂₋₃₀ saturated or unsaturated hydrocarbon chain ofR₂₀, R₂₃ and the 5, 6 or 7 membered unsaturated ring is saturated. 31.The compound of claim 29, wherein said C₂₋₃₀ saturated or unsaturatedhydrocarbon chain of R₂₀, R₂₃ and the 5, 6 or 7 membered unsaturatedring is a straight hydrocarbon chain comprising 6 to 15 carbon atoms.